海獸胃線蟲熱休克蛋白之研究

Abstract

海獸胃線蟲寄生於多種海水魚類和頭足類，廣泛分布於世界各大洋，屬於人畜共通傳染病。本研究自台灣東北海域宜蘭(北緯25度，東經121度) 捕撈250尾花腹鯖(Scomber australasicus)。魚體內採集之線蟲經過初步的型態鑑定後，共檢獲502條海獸胃線蟲第三期幼蟲。利用聚合酶連鎖反應-限制酶片段長度多型性技術(Polymerase chain reaction-restriction fragment length polymorphism) 可精確鑑定物種。 由於 DNA 的多型性使限制酶切位點及片段數目發生改變，藉此可建立簡易的海獸胃線蟲屬內分子分類檢索表。本研究共檢測到六種海獸胃線蟲，分別為Anisakis pegreffii, A. typica, A. paggiae, A. brevispiculata, A. physeteris, 和一種重組種(A. pegreffii × A. simplex sensu stricto)。其中三種海獸胃線蟲為新寄主和新地理紀錄: A. paggiae, A. brevispiculata, A. physeteris。A. pegreffii為優勢種(盛行率= 57.2%)，同時亦是造成海獸胃線蟲症的主要病原之一。最大似然估計（Maximum Likelihood）和近鄰相接法（Neighbor-joining method）建立演化樹之結果形成為兩大演化支，一分支為單系群的A. paggiae, A. brevispiculata, A. physeteris,另一分支為獨立出來的A. typica。為了瞭解海獸胃線蟲在溫度壓力下細胞的反應機制，我們選擇熱休克蛋白90為研究目標。目前已知在其他生物體中，它在細胞過程和壓力環境下扮演重要角色。首先，我們利用cDNA 末端快速擴增技術(Rapid amplification of cDNA ends) 選殖放大五種海獸胃線蟲熱休克蛋白90的全長(重組種除外)。藉由定量即時聚合酶鏈鎖反應分析，可偵測熱休克蛋白90轉錄表現。結果顯示在熱刺激(50˚C)下，熱休克蛋白90 mRNA有逐漸增加的趨勢; 在37˚C影響下，其表現量在處理後的前三小時增加， 之後又降回基準值 ; 在冷刺激(4˚C)下，熱休克蛋白90 mRNA表現量沒有明顯改變。此外，當海獸胃線蟲處理在熱刺激下，其熱休克蛋白90蛋白質表現量會隨處理時間增加呈正相關，藉此推測熱休克蛋白90 mRNA和蛋白質表現量會受熱刺激上調，尤其當海獸胃線蟲處於熱壓力下，熱休克蛋白90 mRNA和蛋白質表現會明顯增加。 為了探討熱休克蛋白70和90在海獸胃線蟲生活史中可能參與的角色，尤其是感染型第三期幼蟲和第四期幼蟲的生活史，分別寄生從冷血魚類轉移到恆溫海洋哺乳類動物或意外宿主人類體中，我們檢測熱休克蛋白70和90在不同發育階段的表現量變化。熱圖和演化樹結果顯示，海獸胃線蟲熱休克蛋白70和90推測的胺基酸序列和其他的線蟲熱休克蛋白70和90呈現高度同源性。海獸胃線蟲熱休克蛋白70和90蛋白質三維結構預測與其他生物具有高度保守性。定量即時聚合酶鏈鎖反應和西方點墨分析顯示這兩種蛋白質在第四期幼蟲表現量顯著高於第三期幼蟲。此外，海獸胃線蟲熱休克蛋白70轉錄表現量高於熱休克蛋白90，推測在不同發育階段，這兩種蛋白可能有不同的調控系統。本研究推測海獸胃線蟲熱休克蛋白70和90蛋白質在生物體處於熱壓力環境下扮演重要角色，同時也可能參與海獸胃線蟲的發育過程。

Anisakis nematodes were found in a variety of marine fishes around the world and they are known to cause anisakiasis in human hosts. Anisakis third-stage larvae (L3, n = 502) were isolated from 250 spotted mackerel Scomber australasicus caught from the coastal waters of Yilan, in northeastern Taiwan (25◦ N, 121◦ E). Anisakis nematodes were pre-identified morphologically and later molecularly by PCR-RFLP. A simple molecular taxonomic key, utilizing RFLP by two restriction enzymes HinfI and HhaI, enabled the differentiation of the genus Anisakis. We obtained six species of the genus Anisakis, A. pegreffii, A. typica, A. paggiae, A. brevispiculata, A. physeteris, and a recombinant genotype between A. pegreffii and A. simplex sensu stricto. Thereby we provide new host and locality records for A. paggiae, A. brevispiculata and A. physeteris. A. pegreffii was determined to be the dominant species (prevalence = 57.2%) and important agent of human anisakiasis. The phylogeny of the Maximum Likelihood and Neighbor Joining trees show two well supported clades: one includes the species of A. pegreffii and the other includes A. paggiae, A. physeteris and A. brevispiculata, while A. typica has basal position to all other Anisakis spp. analyzed. To analyze the cellular response to temperature stress in Anisakis, the heat shock protein 90 (Hsp90) was chosen in the study, as it plays a key role in many cellular processes and responds to stress conditions such as heat or cold shock. The Hsp90 genes of five species (except the recombinant genotype) were cloned by rapid amplification of cDNA ends (RACE). Quantitative RT-PCR showed that Hsp90 transcript levels increased slightly under heat shock (50˚C) treatment, and increased gradually during the first 3 h, and thereafter, returned to its baseline value at 37˚C. Under cold shock (4˚C) treatment, the mRNA expression of Hsp90 did not change significantly. In addition, we found a clear time-dependent Hsp90 protein expression pattern of A. pegreffii exposed to high temperature. Our results suggest that the mRNA and protein expression patterns of Hsp90 are related to the temperature, and are especially significantly increased under heat stress. On the other hand, to elucidate the possible roles of Hsp70 and Hsp90 in the life cycle of the parasitic nematode Anisakis, particularly third- and fourth-stage larvae, which are transferred from cold-blooded fish to warm-blooded marine mammals or accidentally to human hosts, and we examined the expression profiles of Hsp70 and Hsp90 in different developmental stages of A. pegreffii. On heatmap and phylogenetic analysis, ApHsp70 and ApHsp90 shared the highest amino acid sequence identity with other nematodes and formed a monophyletic clade. The three-dimensional (3D) structure prediction of the newly characterized ApHsp70 and known ApHsp90 gene showed highly conserved motifs between A. pegreffii and other species. Quantitative real-time PCR and western blot analysis revealed higher mRNA and protein expression for ApHsp70 and ApHsp90 in fourth- than third-stage larvae, with higher mRNA and protein expression for ApHsp70 than ApHsp90. Our results suggest that ApHsp70 and ApHsp90 may play important roles in Anisakis in response to the thermal stress , and might be important molecules in the development of A. pegreffii, which has implications for its control.